Reversible agglutination mediators

ABSTRACT

Compounds and methods are disclosed for reversibly aggregating particles suspended in a liquid medium. The method comprises combining the liquid medium containing the particles with a polyionic polymer capable of aggregating the particles under conditions suitable for such aggregation. Thereafter, the particles are contacted with a chemical reagent capable of cleaving the polyionic polymer under conditions sufficient to reverse the aggregation. Optionally, magnetic particles are added to the liquid medium in the present method under conditions for non-specific binding and the medium including the aggregates is subjected to a magnetic field gradient to separate the aggregates from the medium. The compounds of the present invention are polyions. The aggregation of the particles is reversible upon contact with chemical agents which cleave at least some of the bonds within the polyionic polymer.

This is a division of pending application Ser. No. 07/051,978, filed May19, 1987, now U.S. Pat. No. 4,812,401.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for reversibly aggregating particlesthat are dispersed in a liquid medium by use of a polyionic polymer toaggregate the particles and a chemical reagent to reverse theaggregation of the particles by cleaving at least some of the bondswithin the polyionic polymer. The invention has particular applicationto separation of cells from biological fluids such as whole blood,lymphatic fluid, urine, cell cultures, etc.

Numerous techniques are known for determining the presence and amount ofan analyte in a sample, such as a biological fluid, for example, bloodor urine. An in vitro assay procedure is the most common of thesetechniques. Many of these techniques involve competitive binding of theanalyte to be determined and a labeled analog of such analyte to bindingsites on a specific receptor, for example, an antibody. Some of thesetechniques involve an aggregation step where the bound or unboundlabeled analog is bound to or associated with a support such as aparticle, which becomes aggregated. The aggregate can then be examinedfor a signal produced in relation to the amount of analyte in thesample.

Several techniques are known for aggregating particles suspended in aliquid medium. For example, the particles may be aggregated by employinga polymer in the medium. In other instances, the particles may beco-aggregated with magnetic particles using a polymer which, forexample, non-specifically binds the particles and the magneticparticles.

Several techniques are known for separating bound and unbound fractions.For example, such techniques include differential migration of the boundand the free fractions, e.g., chromatoelectrophereses, gel filtration,etc., chemical precipitation of the bound or free fraction, e.g., bymeans of organic solvents, salts, acids, etc. followed by filtration orcentrifugation; immunological precipitation of the bound fraction, e.g.by double antibody technique followed by filtration or centrifugation;absorption of the bound or free fraction onto selective sorbing media,e.g., charcoal, silicates, resins, etc.; magnetic separation techniques,and the like.

Magnetic separations generally fall into two general categories. Thereare those separations in which the material to be separated isintrinsically magnetic. The second type involves rendering one or morecomponents of a mixture magnetic by attachment to a magneticallyresponsive entity. In biological separations, for example, materials ofinterest are generally not sufficiently magnetic and therefore, magneticparticles bound to antibodies, lectins and other targeting moleculeshave been used to isolate many of these materials.

The binding of non-magnetic and magnetic particles to each other can beaffected by pH. Therefore, one method that has been suggested forreversing the aggregation of the particles involves altering pH. Bindingcan also be affected by other factors such as ionic strength and thepresence of ionic or non-ionic polymers. In one approach, where theparticles are bound by ionic interactions, the ionic strength isadjusted upwards to facilitate reversal of the coupling of thenon-magnetic particles and the magnetic particles.

2. Description of the Related Art

A method for determining the concentration of substances in biologicalfluids (e.g., drugs, hormones, vitamins and enzymes) whereinmagnetically responsive, permeable, solid, water insoluble,microparticles are employed is disclosed in U.S. Pat. No. 4,115,534.U.S. Pat. No. 4,452,773 discloses magnetic iron-dextran microsphereswhich can be covalently bonded to antibodies, enzymes and otherbiological molecules and used to label and separate cells and otherbiological particles and molecules by means of a magnetic field. Coatedmagnetizeable microparticles, reversible suspensions thereof, andprocesses relating thereto are disclosed in U.S. Pat. No. 4,454,234. Amethod of separating cationic from anionic beads in mixed resin bedsemploying a ferromagnetic material intrinsically incorporated with eachof the ionic beads is described in U.S. Pat. No. 4,523,996. A magneticseparation method utilizing a colloid of magnetic particles is discussedin U.S. Pat. No. 4,526,681. UK Patent Application GB 2,152,664Adiscloses magnetic assay reagents.

SUMMARY OF THE INVENTION

The method of the present invention is directed to the reversibleaggregation of particles suspended in a liquid medium by employing apolyionic polymer to aggregate the particles and by contacting theaggregated particles with a chemical reagent capable of reversing theaggregation by cleaving the polyionic polymer. Where the particles inthe medium are non-magnetic, they may form aggregates with each other,with other non-magnetic particles or with magnetic particles by additionof a polyionic polymer. Where the particles are magnetic, they may formaggregates with each other or with non-magnetic particles by addition ofa polyionic polymer.

The method of the present invention has particular application in theassay of organic or biological analytes, particularly those analytes ofinterest in the analysis of body fluids. Of special interest are assayswhere the analyte is a member of a specific binding pair (sbp) where theanalyte or an sbp member complementary to the analyte is bound, or canbecome bound, to the exterior surface of a particle. If the sbp memberon the particle is not complementary to the analyte, then acomplementary sbp member is also added. The method involves combiningthe particles which are suspended in a liquid medium with an sbp membercomplementary to the analyte or to the sbp member on the surface andadding a polyionic polymer that is capable of non-specificiallyaggregating the particles. After aggregation has occurred, a chemicalreagent that is capable of reversing the aggregation by cleaving atleast some of the bonds of the polyionic polymer is added. Thereafter,the residual specific aggregation of the particles is measured.Normally, the sbp member is detected by virtue of a signal created bythe use of a signal producing system that generates a signal in relationto the amount of the analyte in the sample.

Of special interest are methods such as removing cells from whole blood,where the analyte is a surface component or becomes bound to anon-magnetic particle. In such an instance, the method involvescombining in the medium the sample including non-magnetic particles,such as whole blood, magnetic particles and a polyionic polymer fornon-specifically agglutinating the magnetic particles and thenon-magnetic particles, e.g. the cells. The medium is subjected to amagnetic field gradient to separate the agglutinated cells from bloodplasma. The agglutinated cells are contacted with a chemical reagentunder conditions for reversing the agglutination by at least partialdepolymerization of the polyionic polymer.

The method of the invention provides a way of separating non-magneticparticles from a medium by virtue of non-specifically aggregating suchparticles to magnetic particles by employing a polyionic polymer. Italso provides for reversing the aggregation by employing a chemicalreagent that cleaves at least some of the bonds within the polyionicpolymer.

The present invention also includes novel polyionic polymers includingpolycations of the formula:

    (A).sub.n

wherein A is positively charged and has 4 to 30 atoms other thanhydrogen, wherein the atoms are independently selected from the groupconsisting of carbon, oxygen, phosphorous, nitrogen and sulfur andwherein at least one of the A groups has a cleavable bond; and n is onthe average 5 to 10,000.

Additionally, the invention includes kits for conducting the method ofthe invention and for conducting an assay for determining an analyte ina sample suspected of containing an analyte.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to a method of reversible aggregation ofparticles suspended in a liquid medium. The method involves employing apolyionic polymer for aggregating particles or co-aggregating particleswith each other and reversing the aggregation by employing a chemicalreagent to cleave at least some of the cleavable bonds within thepolyionic polymer. Oftentimes the particle to be separated from theliquid medium is a non-magnetic particle or will be aggregated to, orwill be caused to aggregate to, a non-magnetic particle. On the otherhand, sometimes the particle to be separated is magnetic or will becaused to co-aggregate with a magnetic particle. Therefore, by additionof a polyionic polymer in accordance with the present invention,aggregation will be achieved between non-magnetic particles, betweenmagnetic and non-magnetic particles, or between magnetic particles.

In those cases where the particles to be separated from the liquidmedium are magnetic or where magnetic particles are employed toco-aggregate non-magnetic particles, the aggregated particles areseparated from the medium by the use of a magnetic field gradient. Inthose cases where magnetic particles are not employed, the aggregatesmay be separated from the liquid medium by use of any known methodincluding but not limited to centrifugation, filtration, floatation,distribution between immiscible solvents, absorption onto selectivesorbing media, and the like. The aggregated particles are treated with achemical reagent capable of cleaving at least some of the bonds withinthe polyionic polymer for a time sufficient to reverse the aggregation.

Furthermore, the present invention relates to novel compositions forreversibly aggregating particles suspended in a liquid medium.

The compositions of the present invention are polyionic polymers of theformula:

    (A).sub.n

wherein A is positively charged and has 4 to 30 atoms other thanhydrogen, wherein the atoms are independently selected from the groupconsisting of carbon, oxygen, phosphorous, nitrogen and sulfur, andwherein at least one of said A groups has a cleavable bond; and n is anaverage of 5 to 10,000.

The preferred compositions are polyionic polymers of the formula:##STR1## wherein R₁ and R₂ are the same or different and areindependently selected from the group consisting of aryl, aralkyl,alkyl, alkylene, alkoxyalkyl, substituted aryl, substituted aralkyl,substituted alkyl, substituted alkylene, and substituted alkoxyalkyl,with the proviso that the "alkoxy" portion of the alkoxyalkyl and thesubstituted alkoxyalkyl has from 1 to 6 carbon atoms and the "alkyl"portion in each of the above has from 1 to 6 carbon atoms. B is alinking group containing 2 to 30 atoms other than hydrogen which atomsare independently selected from the group consisting of carbon, oxygen,phosphorous, nitrogen and sulfur, wherein at least one of the B groupshas a cleavable bond which when cleaved provides for a decrease in n,and n is an average of 5 to 10,000. The cleavable bonds are preferablyin disulfides, carboxylic, phosphate, and sulfate esters and amides,carboboranes, siloxanes, vicinal glycols, and the like.

The reagents for cleaving the cleavable bonds include, but are notlimited to, reducing agents such as mercaptans, such asdithioerythritol, hydrolytic enzymes such as pepsin, periodate salts,sulfite, phosphite and borohydride salts, hydrogen peroxide, andfluoride salts.

The present method has wide application in the field of the separationof suspended particles from a medium, particularly for separatingbiological materials such as cells and microorganisms, and in the fieldsof immunoassays and blood typing. The method provides for aggregatingthe particles using a polyionic polymer and for reversing theaggregation by addition of a chemical reagent capable of at leastpartially depolymerizing the polyionic polymer.

The invention provides a method for reversibly aggregating particles,which is more effective than, and eliminates the need for, reversing theionic binding of particles by altering the ionic strength or the pH ofthe medium. The invention also has application to the assay of ananalyte in a sample where a separation step is required.

Before proceeding further with the description of the specificembodiments of the present invention, a number of terms will be defined.

Analyte--the compound or composition to be measured, the material ofinterest. The analyte can be a member of a specific binding pair (sbp)and may be a ligand, which is mono- or polyvalent, usually antigenic orhaptenic, and is a single compound or plurality of compounds which shareat least one common epitopic or determinant site. The analyte can alsobe a component of a particle or can become bound to a particle during anassay. Exemplary of an analyte that is a component of a particle is anantigen on the surface of a cell such as a blood group antigen (A, B,AB, O, D, etc.) or an HLA antigen. Exemplary of an analyte becomingbound to a particle during an assay is an sbp member where acomplementary sbp member is bound to a particle, glycoprotein orglycolipids where a lectin is bound to a particle, antibodies whereprotein A is bound to a particle, and the like. The binding involvedwhen an analyte becomes bound to a particle can be specific ornon-specific, immunological or non-immunological.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

The precise nature of some of the analytes together with numerousexamples thereof are disclosed in U.S. Pat. No. 4,299,916 to Litman, etal., particularly at columns 16 to 23, the disclosure of which isincorporated herein by reference.

For the most part, the polyepitopic ligand analytes employed in thesubject invention will have a molecular weight of at least about 5,000,more usually at least about 10,000. In the poly(amino acid) category,the poly(amino acids) of interest will generally be from about 5,000 to5,000,000 molecular weight, more usually from about 20,000 to 1,000,000molecular weight; among the hormones of interest, the molecular weightswill usually range from about 5,000 to 60,000 molecular weight.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes of interest include drugs, metabolites, pesticides,pollutants, and the like. Included among drugs of interest are thealkaloids. Among the alkaloids are morphine alkaloids, which includesmorphine, codeine, heroin, dextromethorphan, their derivatives andmetabolites; cocaine alkaloids, which include cocaine and benzoylecgonine, their derivatives and metabolites, ergot alkaloids, whichinclude the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids, isoquinoline alkaloids; quinolinealkaloids, which include quinine and quinidine; diterpene alkaloids,their derivatives and metabolites.

The next group of drugs includes steroids, which includes the estrogens,androgens, andreocortical steroids, bile acids, cardiotonic glycosidesand aglycones, which includes digoxin and digoxigenin, saponins andsapogenins, their derivatives and metabolites. Also included are thesteroid mimetic substances, such as diethylstilbestrol.

The next group of drugs is lactams having from 5 to 6 annular members,which include the barbiturates, e.g. phenobarbital and secobarbital,diphenylhydantonin, primidone, ethosuximide, and their metabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes the amphetamines, catecholamines, whichincludes ephedrine, L-dopa, epinephrine, narceine, papaverine, and theirmetabolites.

The next group of drugs is benzheterocyclics which include oxazepam,chlorpromazine, tegretol, imipramine, their derivatives and metabolites,the heterocyclic rings being azepines, diazepines and phenothiazines.

The next group of drugs is purines, which includes theophylline,caffeine, their metabolites and derivatives.

The next group of drugs includes those derived from marijuane, whichincludes cannabinol and tetrahydrocannabinol.

The next group of drugs includes vitamins such as A, B, e.g. B₁₂, C, D,E and K, folic acid and thiamine.

The next group of drugs is prostaglandins, which differ by the degreeand sites of hydroxylation and unsaturation.

The next group of drugs is antibiotics, which include penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, the metabolitesand derivatives.

The next group of drugs is the nucleosides and nucleotides, whichinclude ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidinewith their appropriate sugar and phosphate substituents.

The next group of drugs is miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, amitriptyline,nortriptyline, lidocaine, procaineamide, acetylprocaineamide,propranolol, griseofulvin, valproic acid, butyrophenones,antihistamines, anticholinergic drugs, such as atropine, theirmetabolites and derivatives.

Metabolites related to diseased states include spermine, galactose,phenylpyruvic acid, and porphyrin Type 1.

The next group of drugs is aminoglycosides, such as gentamicin,kanamicin, tobramycin, and amikacin.

Among pesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sulfenamides, theirmetabolites and derivatives.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, DNA, RNA,thyroxine binding globulin, thyroxine binding prealbumin, transcortin,etc.

Ligand analog or analyte analog--a modified ligand or ligand surrogateor modified analyte or analyte surrogate which can compete with theanalogous ligand or analyte for a receptor, the modification providingmeans to join a ligand analog or analyte analog to another molecule. Theligand analog or analyte analog will usually differ from the ligand oranalyte by more than replacement of a hydrogen with a bond which linksthe ligand analog or analyte analog to a hub or label, but need not. Theterm ligand surrogate or analyte surrogate refers to a compound havingthe capability of specifically binding a receptor complementary to theligand or analyte. Thus, the ligand surrogate or analyte surrogate canbind to the receptor in a manner similar to the ligand or analyte. Thesurrogate could be, for example, an antibody directed against theidiotype of an antibody to the ligand or analyte.

Poly(ligand analog)--a plurality of ligand analogs joined togethercovalently, normally to a hub nucleus. The hub nucleus is apolyfunctional material, normally polymeric, usually having a pluralityof functional groups, e.g., hydroxyl, amino, mercapto, ethylenic, etc.as sites for linking. The hub nucleus may be water soluble or insoluble,preferably water soluble, and will normally be at least about 30,000molecular weight and may be 10 million or more molecular weight.Illustrative hub nuclei include polysaccharides, polypeptides (includingproteins), nucleic acids, anion exchange resins, and the like. Waterinsoluble hub nuclei can also include walls of containers, e.g. glass orplastic, glass beads, addition and condensation polymers, Sephadex andAgarose beads and the like.

Member of a specific binding pair ("sbp member")--one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones-hormone receptors, nucleic acidduplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like are notimmunological pairs but are included in the invention.

Ligand-any organic compound for which a receptor naturally exists or canbe prepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component Clq, and the like.

Non-magnetic particles--diamagnetic or paramagnetic particles usuallywith a magnetic susceptibility (χ) of less than 1×10⁻⁵ emu/Oecm³. Thenon-magnetic particles are generally at least about 0.02 microns and notmore than about 100 microns, usually at least about 0.05 microns andless than about 20 microns, preferably from about 0.3 to 10 micronsdiameter. The non-magnetic particle may be organic or inorganic,swellable or non-swellable, porous or non-porous, preferably of adensity approximating water, generally from about 0.7 to about 1.5 g/ml,and composed of material that can be transparent, partially transparent,or opaque. Usually the non-magnetic particles will have a charge, eitherpositive or negative, and may have sbp members on their surface.Normally, the non-magnetic particles will be biologic materials such ascells and microorganisms, e.g., erythrocytes, leukocytes, lymphocytes,hybridomas, streptococcus, staphylococcus aureus, E. coli, viruses, andthe like. The non-magnetic particles can also be particles comprised oforganic and inorganic polymers, liposomes, latex particles, phospholipidvesicles, chylomicrons, lipoproteins, and the like.

The polymers will normally be either addition or condensation polymers.Non-magnetic particles derived therefrom will be readily dispersible inthe assay medium and may be adsorptive or functionalizable so as tobind, either directly or indirectly, an sbp member or a magneticparticle.

Frequently, the non-magnetic particles will be an analyte, be bound toan analyte, or will become bound to an analyte during an assay. Thenon-magnetic particles not initially bound to the analyte can be derivedfrom naturally occurring materials, naturally occurring materials whichare synthetically modified and synthetic materials. Among organicpolymers of particular interest are polysaccharides, particularlycross-linked polysaccharides, such a agarose, which is availabe asSepharose, dextran, available as Sephadex and Sephacryl, cellulose,starch, and the like; addition polymers, such as polystyrene, polyvinylalcohol, homopolymers and copolymers of derivatives of acrylate andmethacrylate, particularly esters and amides having free hydroxylfunctionalities, and the like.

The non-magnetic particles for use in assays will usually bepolyfunctional and will have bound to or be capable of specificnon-covalent binding to an sbp member, such as antibodies, avidin,biotin, lectins, protein A, and the like. A wide variety of functionalgroups are available or can be incorporated. Functional groups includecarboxylic acids, aldehydes, amino groups, cyano groups, ethylenegroups, hydroxyl groups, mercapto groups and the like. The manner oflinking a wide variety of compounds of particles is well known and isamply illustrated in the literature. See for example Cautrecasas, J.Biol. Chem., 245 3059 (1970). The length of a linking group may varywidely, depending upon the nature of the compound being linked, theeffect of the distance between the compound being linked and theparticle on the binding of sbp members and the analyte and the like.

The non-magnetic particle will normally have an electronic charge,either positive or negative. The particle can be inherently charged orcan be treated chemically or physically to introduce a charge. Forexample, groups such as carboxyl, sulfonate, phosphate, amino, and thelike can be chemically bound to or formed on the particles by techniquesknown in the art. Cells are normally negatively charged due to thepresence of sialic acid residues on the cell surface. Latex particlescan be positively or negatively charged but normally will have anegative charge as a result of the introduction of functional groups orabsorption of charged polymers such as polypeptides, proteins,polyacrylate, and the like.

The non-magnetic particles can be fluorescent or non-fluorescent,usually non-fluorescent, but when fluorescent can be either fluorescentdirectly or by virtue of fluorescent compounds or fluorescers bound tothe particle in conventional ways. The fluorescers will usually bedissolved in or bound covalently or non-covalently to the non-magneticparticle and will frequently be substantially uniformly bound throughthe particle. Fluoresceinated latex particles are taught in U.S. Pat.No. 3,853,987 and are available commercially as Covaspheres fromCovalent Technology Corp.

The fluorescers of interest will generally emit light at a wavelengthabove 350 nm, usually above 400 nm and preferably above 450 nm.Desirably, the fluorescers have a high quantum efficiency, a largeStokes shift and are chemically stable under the conditions of theirconjugation and use. The term fluorescer is intended to includesubstances that emit light upon activation by electromagnetic radiationor chemical activation and includes fluorescent and phosphorescentsubstances, scintillators, and chemiluminescent substances.

Fluorescers of interest fall into a variety of categories having certainprimary functionalities. These primary functionalities include 1- and2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p'-diaminostilbenes, imines,anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene,7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate,strophanthidin, porphyrins, triarylmethanes, flavin and rare earthchelates oxides and salts. Exemplary fluorescers are enumerated in U.S.Pat. No. 4,318,707, columns 7 and 8, the disclosure of which isincorporated herein by reference. Squaraine dyes described in U.S.patent application Ser. No. 773,401, filed Sept. 6, 1985 (the relevantdisclosure of which is incorporated by reference) are also useful asfluorescers.

Additionally, light absorbent non-magnetic particles can be employedwhich are solid insoluble particles of at least about 10 nm in diameter.

Many different types of particles may be employed. Of particularinterest are carbon particles, such as charcoal, lamp black, graphite,colloidal carbon and the like. Besides carbon particles metal sols mayalso find use, particularly of the noble metals, gold, silver, andplatinum.

Label--A member of the signal producing system that is conjugated to ansbp member. The label can be isotopic or non-isotopic, usuallynon-isotopic, including catalysts such as an enzyme, a chromogen such asa fluorescer, dye or chemiluminescer, a radioactive substance, aparticle, and so forth.

Signal Producing System--The signal producing system may have one ormore components, at least one component being a label. The signalproducing system generates a signal that relates to the presence oramount of analyte in a sample. The signal producing system includes allof the reagents required to produce a measurable signal. When the labelis not conjugated to an sbp member analogous to the analyte, the labelis normally bound to an sbp member complementary to an sbp member thatis analogous to the analyte. Other components of the signal producingsystem can include substrates, enhancers, activators, chemiluminiscentcompounds, cofactors, inhibitors, scavengers, metal ions, specificbinding substances required for binding of signal generating substances,and the like. Other components of the signal producing system may becoenzymes, substances that react with enzymic products, other enzymesand catalysts, and the like. The signal producing system provides asignal detectable by external means, preferably by measurement of thedegree of aggregation of particles or by use of electromagneticradiation, desirably by visual examination. For the most part, thesignal producing system will involve particles, such as fluorescentparticles or other light absorbing particles, a chromophoric substrateand enzyme, where chromophoric substrates are enzymatically converted todyes which absorb light in the ultraviolet or visible region, phosphors,fluorescers or chemiluminescers.

The signal producing system can include at least one catalyst, usuallyan enzyme, and at least one substrate and may include two or morecatalysts and a plurality of substrates, and may include a combinationof enzymes, where the substrate of one enzyme is the product of theother enzyme. The operation of the signal producing system is to producea product which provides a detectable signal related to the amount ofanalyte in the sample.

A large number of enzymes and coenzymes useful in a signal producingsystem are indicated in U.S. Pat. No. 4,275,149, columns 19 to 23, andU.S. Pat. No. 4,318,980, columns 10 to 14, which disclosures areincorporated herein by reference. A number of enzyme combinations areset forth in U.S. Pat. No. 4,275,149, columns 23 to 28, whichcombinations can find use in the subject invention. This disclosure isincorporated herein by reference.

Of particular interest are enzymes which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference. When a single enzyme is used as a label,other enzymes may find use such as hydrolases, transferases, andoxidoreductases, preferably hydrolases such as alkaline phosphatase andβ-galactosidase. Alternatively, luciferases may be used such as fireflyluciferase and bacterial luciferase.

Illustrative coenzymes which find use include NAD[H]; NADP[H], pyridoxalphosphate; FAD[H]; FMN[H], etc., usually coenzymes involving cyclingreactions, see particularly U.S. Pat. No. 4,318,980.

The product of the enzyme reaction will usually be a dye or fluorescer.A large number of illustrative fluorescers are indicated in U.S. Pat.No. 4,275,149, columns 30 and 31, which disclosure is incorporatedherein by reference.

Magnetic particles--particles that are intrinsically magneticallyresponsive or have been rendered magnetic by, for example, attachment toa magnetically responsive substance or by incorporation of suchsubstance into the particles. The magnetic particles can beparamagnetic, ferromagnetic, or superparamagnetic, usually paramagneticand will have magnetic susceptibilities (χ) of at least 5×10⁻⁵emu/Oecm³, usually at least 4×10⁻⁴ emu/Oecm3. The diameter of theparticles should be small, generally in the range from about 5 nm to 50microns, preferably from about 20 nm to 5 microns, more preferably fromabout 50 mn to 1 micron, frequently colloidal.

Exemplary of the magnetic component of particles that are intrinsicallymagnetic or magnetically responsive are complex salts and oxides,borides, and sulfides of iron, cobalt, nickel and rare earth elementshaving high magnetic susceptibility, e.g. hematite, ferrite. Themagnetic component of other such particles includes pure metals oralloys comprising one or more of these elements.

For the most part the magnetic particles will contain a core of themagnetic component with surface functional groups such as hydroxyl,silicate, carboxylate, sulfate, amino, phosphate and the like.Frequently, an additional surface coating will be employed that iscovalently or non-covalently bound to the surface. The surface coatingcan be an anionic or cationic detergent, usually anionic; or the coatingcan be a protein such as albumin, immunoglobulin, avidin, fetuin or thelike; or it can be a carbohydrate such as dextran, chitosan, amylose andthe like, or combinations or these substances in their native form orfunctionalized so as to control their charge and hydrophilicity.Alternatively, the particles can be coated with other amphiphilicsubstances such as lipopolysaccharides, octyl glucoside, etc.

Alternatively, the magnetic component can be incorporated into aparticle such as, for example, impregnating the substance in a polymericmatrix. For a more in-depth discussion of the preparation of magneticparticles by this method, see Whitesides, et al. (1983) Trends inBiotechnology, 1(5): 144-148 and references cited therein.

In those cases wherein it is desirable to use small magnetic particles,magnetic particles of less than a hundred nanometers in diameter can bemade by precipitating iron oxides in the presence or absence of acoating such as a polysaccharide or protein. Magnetic particles of a fewmicrons diameter can also be made by a ball milling process and removingmaterial that is not in the size range of interest. Typically, magneticparticles formed by this latter process are quite polydisperse. Metaloxide suspensions that are generally monodisperse can be prepared bycareful control of pH, temperature and concentrations during theprecipitation process. Coating the magnetic particles withmacromolecules can increase their colloidal stability. This can be doneby direct adsorption of high molecular weight polymers or byfunctionalizing the surface of the particle and then bindingmacromolecules to the functional groups. Emulsion polymerization andgrafting techniques provide a means for coating magnetic particles withpolymers.

In general, the magnetic particle that is best for a given task will bedetermined primarily by the size and properties of the particles to beseparated. For immunoassays or the isolation of cells, the magneticparticles preferably should be readily suspendable, form stable,preferably colloidal, suspensions, and have high magneticsusceptibility. Where an sbp member is bound to the surface, its abilityto bind to a complementary sbp should be retained and should be stablewith time.

Small (<100 nm) magnetic particles are advantageously used inimmunoassays and cell separation procedures. These particles preferablyhave a homogenous core of metal, metal oxide or other metal compound.When colloidally stable, small particles can be suspended for longperiods of time. Their large surface to volume ratio and relativelyhigher rates of diffusion enable them to quickly bind molecules andparticles that are dispersed in the medium. Small magnetic particles arealso less susceptible than large magnetic particles to aggregation dueto residual magnetic moments after they have been exposed to a largeapplied magnetic field. Also, methods are known for colloidallystabilizing such small particles.

Magnetic particles of an intermediate size (100-1000 nm) can besuspended readily and require a lower surface charge density to preventspontaneous aggregation than do smaller particles. Magnetic particles ofthis size range can be created by emulsion polymerization and have theadvantage of a surface that is easily modified whether by grafting orthe covalent bonding of macromolecules to their surface. However, theyremain suspended for shorter times and their lower surface to volumeratio decreases the rate of binding to the substance to be separated.

Magnetic fluid--a colloidal suspension of magnetic particles in a liquidcarrier that are not readily separated by a magnetic field. Theresulting liquid has the bulk properties of a magnetic material. Thefluid becomes spontaneously magnetized in the presence of an externalmagnetic field. The liquid also acts as a fluid and is capable ofassuming the shape of its container, of flowing, and of moving aroundobstacles. Exemplary of a magnetic fluid is a ferrofluid where thesuspended particles are ferromagnetic particles (see, for example,Rosenweig, supra, and U.S. Pat. No. 4,019,994, the disclosure of whichis incorporated herein by reference, and Khalafolla, et al. (1980) IEEETransactions on Magnetics, MAG-16:178-183).

The colloidal magnetic particles can be coated with protein material,e.g., a serum protein such as albumin, gammaglobulin, etc., and thelike. The colloidal magnetic particles can be mixed with an aqueousbuffered solution of protein to prepare the protein-coated colloidalmagnetic particles. The coating of the magnetic particles with proteincan be accomplished by physical (e.g., absorption) or chemical binding.

Non-specific binding--non-covalent binding between particles that isrelatively independent of specific surface structures. Such non-specificbinding will usually result from electrostatic interactions betweenoppositely charged particles or between particles having the same chargewhere a polyionic reagent having a charge opposite thereto is employed.Non-specific binding may also result from hydrophobic interactionsbetween particles.

Polyionic polymer--a compound, composition, or material, eitherinorganic or organic, naturally occurring or synthetic, having at leastfive of the same charge, either polyanionic or polycationic, preferablyat least ten of the same charge; e.g., a polyelectrolyte.

The polyionic polymer of the present invention is capable of aggregatingparticles in a liquid medium and has bonds capable of being cleaved by achemical reagent to reverse the aggregation of the particles. Examplesof cleavable bonds in the polyionic polymer are disulfides, carboxylic,phosphate and sulfate esters, amides, carboboranes, siloxanes, vicinalglycols, and the like.

Polyionic polymers useable in the present invention incude polymershaving the following formula:

    (A).sub.n

wherein A is positively charged and has 4 to 30 atoms other thanhydrogen, where the atoms are independently selected from the groupconsisting of carbon, oxygen, phosphorous, nitrogen and sulfur, and atleast one of the A groups has a cleavable bond; and n is an average of 5to 10,000.

A preferred polycationic polymer of the present invention has thefollowing structure: ##STR2## wherein R₁ and R₂ are the same ordifferent and are independently selected from the group consisting ofaryl, aralkyl, alkyl, alkylene, alkoxyalkyl, substituted aryl,substituted aralkyl, substituted alkyl, substituted alkylene, andsubstituted alkoxyalkyl, with the proviso that the "alkoxy" portion ofthe alkoxyalkyl and the substituted alkoxyalkyl has from 1 to 6 carbonatoms and the "alkyl" in each of the above has from 1 to 6 carbon atoms.

B is a linking group containing 2 to 30 atoms other than hydrogen whichatoms are independently selected from the group consisting of carbon,oxygen, phosphorous, nitrogen and sulfur, and at least one of said Bgroups has a cleavable bond; and n is an average of 5 to 10,000.Preferably the cleavable bonds include disulfides or glycols.

Another preferred polycationic polymer has the aforementioned structurewhere R₁ and R₂ are the same or different and are independently selectedfrom the group consisting of alkyl, alkylene and alkoxyalkyl with theproviso that the "alkoxy" portion of the alkoxyalkyl has from 1 to 6carbon atoms and the "alkyl" portion of the alkyl, alkylene andalkoxyalkyl has from 1 to 6 carbon atoms; B is independently selectedfrom a group consisting of polyalkylene, or 0,0' bis-alkylenylpolyether,bis-alkylenyl disulfide and bis-alkylenyl ethylene glycol wherein atleast one of the B groups has a disulfide or glycol group; and n is anaverage of 5 to 10,000.

Another preferred polycationic polymer has the aforementioned structurewherein R₁ and R₂ are the same or different and are independentlyselected from the group consisting of alkyl groups of from 1 to 4 carbonatoms; B is independently selected from a group consisting of--(CH₂)_(a) --(S--S)_(b) --(CH₂)_(c) -- where b is 0 or 1, a and c arefrom 2 to 8 when b is 1, and a and c are from 2 to 10 when b is 0, withthe proviso that in at least one of the B groups b is 1; and n is anaverage of 10 to 10,000, and preferably n is an average of 10 to 20.

An additional preferred polycation has the aforementioned structurewhere R₁ and R₂ are methyl; B is (CH₂)_(m) [CH(OH)]₂ (CH₂)_(p), m and pare 1 to 8; and n is an average of 10 to 100. Another preferredpolycation has the aforementioned structure wherein R₁ and R₂ aremethyl; B is --(CH₂)_(a) --SS--(CH₂)_(c) --, where a and c are 3 to 5;and n is an average of 10 to 20.

Reversing agent--a chemical compound, composition, or material, eithernaturally occurring or synthetic, organic or inorganic, capable ofreversing the aggregation of particles by at least partialdepolymerization of the polyionic polymer. The reversing or cleavingagent acts upon the bonds of the polyionic polymer and cleaves on theaverage at least one bond per polymer, preferably at least 2 bonds perpolymer.

The choice of specific reversing reagents depends on the cleavable bondswithin the polyionic polymer. The following reversing agents areprovided by way of example and are not meant to be a limitation on thescope of the present invention. Generally, the reversing agents areselected from the group consisting of hydrolytic enzymes, such aspepsin, trypsin, chymotrypsin, phosphodiesterase, and the like;mercaptans, for example mercaptoethanol, dithioerythritol, andglutathione, and the like; sulfite, halide, phosphite, periodate andborohydride salts; and peroxides, such as hydrogen peroxide.

As has been pointed out and by way of example, the specific reversingreagent chosen is dependent on the bonds that will be cleaved. Forexample, when the cleavable bond connects two sulfur atoms, the reagentselected will generally be a reducing agent such as, for example,dithioerythritol, and the like. Where the cleavable bond is a peptidebond, the reversing reagent may be trypsin, and the like. In cases wherethe cleavable bond connects the carbon atoms of a vicinal glycol, thereversing agent may be periodate, such as sodium periodate, and thelike. Where the cleavable bond connects the carbon and oxygen atoms ofan ester, the reversing agent may be chymotrypsin, sodium hydroxide andthe like. Where the bond is a phosphate ester the reversing agent may bephosphodiesterase, and where the bond is carboborane the reversing agentmay be hydrogen peroxide. Where the bond is siloxane the agent may be afluoride salt.

Ancillary materials--various ancillary materials will frequently beemployed in an assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers for the assay medium and the assay components.Frequently, in addition to these additives, additional proteins may beincluded, such as albumins, or surfactants, particularly non-ionicsurfactants, binding enhances, e.g., polyalkylene glycols, and the like.

As mentioned above, the present invention involves a method forreversibly aggregating particles dispersed or suspended in a liquidmedium. The method comprises combining the liquid medium with apolyionic polymer. The polyionic polymers of the present invention arecapable of aggregating the particles and are also capable of beingcleaved so as to reverse the aggregation. The liquid medium containingthe particles and the polyionic polymer is allowed to incubate for atime sufficient for aggregation of the particles to occur. Thereafterthe aggregated particles are contacted with a chemical reagent that iscapable of cleaving the polyionic polymer, under conditions, e.g., time,temperature concentration of reagent and the like to reverse theaggregation.

The particles to be separated will frequently be non-magnetic particles,or will be bound to non-magnetic particles, for example, red bloodcells. In such cases, it is usually convenient to employ magneticparticles. Generally, coaggregation of the non-magnetic particles withmagnetic particles is obtained by including magnetic particles and apolyionic polymer in the liquid medium. Where non-magnetic particles andmagnetic particles are employed having the same charge, a polyionicpolymer having an opposing charge is selected. After the medium isallowed to incubate for a time sufficient to form an aggregate, themedium is subjected to a magnetic field gradient to separate theaggregated particles from the medium. After the particles are separatedfrom the medium the aggregation of the particles is reversed. Incarrying out the method of the invention, the aggregation is reversed bycontacting the particles with the reversing agent.

Moderate temperatures are normally employed for carrying out the methodof the present invention and usually constant temperatures are usedduring the period for conducting the method. Generally, the temperatureswill be chosen to promote aggregation or coaggregation of the particlesby binding of the particles to the polyionic polymer. The temperaturefor the aggregation of the particles, particularly involving an assay,will generally range from about 0° to 50° C., more usually from about15° to 40° C. Again, contacting the aggregated particles with thereversing agent, a temperature that promotes reversal of the aggregationor coaggregation of the particles by cleaving the polyionic polymer canbe chosen. The temperature for the reversal of aggregation, particularlyinvolving an assay, will generally range from about 0° to 50° C., moreusually from about 15° to 40° C.

Where non-magnetic particles are to be separated from a medium, theconcentration of the non-magnetic particles can vary widely dependingupon the need. For example, in separation of cells from blood, the cellvolume may represent fifty percent of the total volume of the blood. Bycontrast, it may be desired to separate as few as 1,000 bacteria/ml froma sample of water. When it is necessary to obtain non-magnetic particlesthat are relatively free of the medium as in an assay, usually the totalvolume of the non-magnetic particles should be less than five percent ofthe medium. In an assay where the analyte is a component of a particleor becomes bound to a particle, the analyte will generally vary fromabout 10⁻⁴ to 10⁻¹⁴ M, more usually from about 10⁻⁶ to 10⁻¹² M.

In those instances where magnetic particles are added to the liquidmedium in which particles of interest are suspended, the concentrationof the magnetic particles added to the medium will depend on thequantity of particles in the medium that are to be separated, and therate of separation that is desired, and the like. The concentration ofmagnetic particles will also depend on the magnetic field gradient andfield strength, the magnetic susceptibility of the magnetic particlesand the like. In general, the higher the concentration of magneticparticles added to aggregate the particles suspended in the liquidmedium the more efficient and rapid will be the separation; however, toohigh a concentration can cause excessive entrainment of the medium. Theconcentration of magnetic particles added to the medium is normallydetermined empirically and will generally vary from about 0.1 to 1000μg/ml, more usually from about 0.5 to 200 μg/ml, frequently from about 1to 50 μg/ml.

In those instances where non-magnetic particles other than naturalparticles associated with the analyte are added to the medium in whichparticles of interest are suspended, their concentration will depend onnumerous factors such as particle size and surface area, concentrationof the particles in the medium, desired rate of separation and the like.In general, the concentration of non-magnetic particles added to themedium will normally be determined emperiodically and will generallyvary from about 0.01 to 100 μg/ml, more usually from about 0.1 to 20μg/ml. The concentration of non-magnetic particles will also depend ontemperature, solubility, viscosity, the method of separation that willbe employed and the like.

While the concentrations of the various reagents will generally bedetermined by the concentration range of interest of the particles to beaggregated and disaggregated with or without intervening separationprocedures or the concentration range of the analyte in an assay, thefinal concentration of each of the reagents will normally be determinedempirically to optimize the rate and extent of aggregation,disaggregation and separation of the particles as the case may be and,in the case of an assay, the sensitivity and specificity of the assayover the range of interest. Other factors to be considered includenon-specific and specific binding effects, desired rate of reaction,temperature, solubility, viscosity and the like.

The polyionic polymer for aggregating the particles is included in theliquid medium. As has been mentioned above, the polyionic polymeremployed in the invention is capable of being at least partiallydepolymerized by a reversing reagent. The polyionic polymer has a chargeopposite to that of the particles. The amount of polyionic polymer addedshould be sufficient so that substantially all of the particles becomeaggregated or coaggregated. This concentration may be determinedempirically. Generally, the polyionic polymer will have a concentrationin the liquid medium sufficient to provide a number of ions associatedwith the polymer and equal to the total number of charges of oppositesign on all the particles in the medium. In an assay, as discussed abovewherein the analyte varies from about 10⁻⁴ to 10⁻¹⁴ M, the polyionicpolymer will have a concentration of about 10 nm to 1 mm, more usuallyabout 100 nm to 100 nm.

In an assay, the aqueous medium can also contain one or more members ofa signal producing system. As mentioned above, the concentration of thevarious members of the signal producing system will vary and bedependent upon the concentration range of interest of the analyte andthe type of measurement or assay involved. As a general point, theconcentration of the various members of the signal producing system willbe selected to optimize the signal produced in relation to theconcentration range of interest of the analyte.

Non-specific aggregation will occur essentially instantaneously, and itis usually sufficient to allow the mixture to stand for 60 seconds,frequently less than 15 seconds. Where specific binding is required, theliquid medium is held for a period of time sufficient for binding tooccur. Normally, this requires 0.5 to 120 minutes, more frequently 1 to60 minutes.

In those instances where magnetic particles are employed, the magneticfield is applied preferably immediately after mixing. The extent ofbinding between the particles and the magnetic particles or betweenmagnetic particles controls the efficiency of the magnetic separation.The application of a magnetic field to the medium to separate theparticles from the medium can be carried out in a conventional mannerthat provides for a high magnetic field gradient. Normally, the methodis conducted in a container made of non-magnetic material, for example,glass or plastic. In applying the magnetic field, the reaction containercan be placed in close proximity to an electromagnet or permanentmagnet, preferably permanent, which has a geometry to maximize the fieldintensity and gradient within the suspension. The higher the strength ofthe magnetic field and the higher the gradient, the faster theseparation. Normally, it will be convenient to carry out the separationin a tube of diameter from about 2 to 50 mm, preferably from about 3 to15 mm, with one or more permanent magnets mounted as close to the tubeas practical to provide field strengths of at least about 200 Oe andpreferably at least about 1 KOe with magnetic field gradients usually atleast about 20 KOe/cm. The magnetic field is applied for a sufficientperiod of time to provide the desired degree of separation of theparticles from the medium. Depending on the geometry, field strength,magnetic susceptibility of the particle and the like, the magnetic fieldis applied for a period of about 2 seconds to 1 hour, preferably about 5seconds to 60 seconds.

Once the particles have been concentrated to one part of the container,the suspending liquid medium can be separated from the particles by anyconvenient means such as, for example, decantation, pipetting, and thelike.

The present invention also has application in those instances wherenon-magnetic particles are to be separated from a liquid medium withoutemploying magnetic particles. In such cases the aggregated non-magneticparticles may be separated from the medium by any convenient method.Such methods, include by way of example but not limitation, settling,centrifugation, floatation, distribution between immiscible solvents,filtration, absorption onto selective sorbing media, e.g., charcoal,silicates, resins, etc.; and the like.

The particles separated from the liquid medium are treated to reversethe aggregation of the particles. Generally, the particles are suspendedin a liquid medium with a chemical reagent capable of reversing theaggregation.

The reversal of the aggregation of the particles and the coaggregationof the particles and the magnetic particles is effected by cleaving atleast some of the bonds within the polyionic polymer. Therefore, thereversing agent chosen will be dependent on the polyionic polymeremployed. Additionally, it is important to choose the reversing agentwith regard to the nature of the particles in the aggregate so as tominimize or avoid damage to the particles after the reversal of theaggregation. The reversing agent selected will at least partiallydepolymerize the polyionic polymer employed to aggregate or coaggregatethe particles.

The concentration of the reversing agent should be sufficient to resultin substantial or complete reversal of the aggregation or coaggregationof the particles. The concentration of the reversing agent is generallydependent upon the nature of bonds of the polyionic polymer that arebeing cleaved. Generally, the concentration of the reversing agent willbe at least equal to the concentration of the bonds to be cleaved,preferably at least ten times the concentration of the bonds, morepreferably at least one hundred times the concentration of the bonds tobe cleaved.

Depending on the strength and number of the bonds to be cleaved, and thenature and concentration of the reversing agent, the temperature andtime needed for reversal of aggregation will vary. Generally, thetemperature will range from about 0° to 45° C., more usually from about15° to 40° C. Likewise, the time needed to reverse aggregation willrange from about 0 to 45 minutes, usually from 15 to 40 minutes.

Once the particles have been separated from the aggregate, they may beused as desired. For example, in an assay the separated particles can beexamined for the presence of a detectable signal in relation to theamount of an analyte in the sample. The separated particles can be cellswhich can be used as desired. For example, the separated particles canbe red blood cells, test cells, and the like.

In a preferred embodiment of the invention, the magnetic particles areprovided as a magnetic liquid, e.g., ferrofluid. The particles to beseparated are combined with the magnetic liquid.

An important application of the present method is the removal of cellsfrom a sample containing cells such as, for example, removal of redblood cells from whole blood. In the method, using whole blood by way ofexample and not by way of limitation, a whole blood sample is combinedin a liquid medium with charged magnetic particles under conditions fornon-specific binding of the magnetic particles to the cells in thepresence of a polyionic polymer. The cells will usually have a negativecharge by virtue of sialic acid residues or the like on the surface ofthe cells. Generally, the magnetic particles have a negative charge. Apolycationic polymer capable of aggregating the particles and alsohaving cleavable bonds such that reversal of said aggregation may beeffected is included in the medium to provide conditions fornon-specific binding between the cells and the magnetic particles.Polycationic reagents of the present invention described in detailherein are useful in this method.

Next, the medium can be subjected to a magnetic field gradient toseparate the aggregated cells from the medium. Application of themagnetic field results in concentration of the cell-magnetic particleaggregate to one portion of the container, which permits its removal ofthe residual cell-free medium by, for example, decantation, pipetting,etc.

The separated cell-magnetic particle aggregate can then be treated torelease the cells from the aggregate as described above. The reversingreagent selected will be dependent on the nature of the bonds of thepolycationic polymer as indicated above.

The present method provides particular advantages for automated bloodtyping procedures by providing a way to prepare blood plasma withoutcentrifugation. It is also useful in the Coombs antiglobulin test whereimmunoglobulin-containing plasma is first combined with test cells andmust then be fully removed in order to determine if antibodies from theplasma have bound to the cells. In this procedure magnetic particles anda polyionic polymer which acts as non-specific aggregating agent areadded to the mixture of plasma and test cells and the subsequentseparated cells are resuspended with the help of a reversing agent whichcleaves the bonds of the polyionic polymer. Moreover, the present methodcan be employed in immunoassays wherein an spb member is bound to aparticle and it is desired to separate and wash the particles withoutcentrifugation; the particles can be magnetic or non-magnetic.

The present invention has application in general to assays for ananalyte in a sample suspected of containing the analyte. The analyte isan spb member. In the assay the sample is combined in an assay mediumwith an spb member complementary to the analyte wherein at least one ofthe analyte or the complementary spb member is associated with thesurface of a non-magnetic particle, usually a cell, such as anerythrocyte, a latex particle, or a magnetic particle. Charged magneticparticles are also combined with the medium under conditions fornon-specific binding and aggregation of the particles by using apolyionic polymer to cause non-specific binding between the particlesand magnetic particles. The present invention offers the improvement ofreversing the aggregation using a chemical means to cleave the bonds ofthe aggregating polymer.

The assay will normally involve a signal producing system for producinga detectable signal in relation to the amount of analyte in the sample.The signal producing system usually includes a labeled sbp member. Themedium may be further combined with none, one or more members of thesignal producing system. Where magnetic particles are employed, themedium is subjected to a magnetic field gradient to separate aggregatescomprising the magnetic particles from the medium. A chemical reagentcapable of cleaving the polyionic polymer is added to the separatedparticles. The residual specific aggregation of the particles can thenbe measured. Such a determination can require the addition of anyremaining members of the signal producing system not added above.

As a matter of convenience, the reagents for aggregating the particlesand reversing the aggregation can be provided in a kit in packagecombination in predetermined amounts for aggregation and reversal ofaggregation of a predetermined analyte. The kit can comprise a) apolymeric reagent for aggregating particles in a liquid medium and b) areversing agent for reversing the aggregation of the particles bycleaving bonds within the polymeric reagent. Additionally, the kit canalso include magnetic particles and/or ancillary agents as necessary.

As a matter of convenience, the reagents for conducting an assay can beprovided in a kit in packaged combination in predetermined amounts foruse in assaying for an analyte. The kit can comprise (a) an sbp membercomplementary to the analyte, (b) an sbp member bound to a chargedparticle if neither the analyte nor the complementary sbp member isbound to a charged particle, (c) charged magnetic particles if thecharged particle is not magnetic, and (d) a polymeric reagent fornon-specifically binding the magnetic particles or the magneticparticles and the non-magnetic particles wherein the polymeric reagentis capable of being cleaved by a chemical reagent whereby saidnonspecific binding is reversed. Additionally, the kit can also includethe chemical reagent for reversing the nonspecific binding and reagentsfor generating a signal in relation to the amount of analyte in thesample. Ancillary agents can be included as necessary for the particularassay.

EXAMPLES

The invention is described further by the following illustrativeexamples. All parts and percentages herein are by volume unlessotherwise indicated. Temperatures are in degrees Centigrade (°C.). NMRspectra were run on a Varian T60 spectrometer. UV spectra were run on aCary 210 spectrophotometer. Other spectrometers or spectrophotometersmay be utilized.

Before describing the Examples a number of terms will be defined.

DEFINITIONS

Latex beads--0.297μ acrylated polystyrene latex, surfactant free, fromIDC of Portland, Oregon.

LISS--0.23M glycine, 0.029M NaCl, 0.0017M KH₂ PO₄, and 0.0013M Na₂ HPO₄,pH 6.7

KOH--potassium hydroxide

H₂ O₂ --hydrogen peroxide

Polybrene--obtained from Sigma Chemical Company, St. Louis, Mo.

DMSO--dimethylsulfoxide

DTE--dithioerythritol

Buffer--0.02M ammonium carbonate, pH 7.

EXAMPLE 1 Preparation of Disulfide of 2-Dimethylaminoethanethiol (1)

A homogeneous solution of 2-dimethylaminoethanethiol hydrochloride (14.2g, 100 mmol) in methanol (50 ml) was stirred and cooled to 0° C. To thestirring chilled solution was added KOH (101 mmol) followed by slowaddition of H₂ O₂ (49.9 mmol). After 15 minutes, the methanol wasevaporated, and the resultant mixture was extracted three times withether. The ether extract was dried (Na₂ SO₄), filtered, and evaporatedto give 10.1 g of a pale yellow oil. Distillation of the oil under highvacuum gave 10 g of the disulfide (1) as a colorless liquid. ##STR3##

EXAMPLE 2 Pseudobrene (ψ-Brene) was prepared using the two methods (Aand B) below:

Method A: Diaminedisulfide (1) (1.043 g, 5 mmol) as prepared in Example1 and 1,3-dibromopropane (1.03 g, 5 mmol) were added to about 4 ml ofDMSO. The reaction was stoppered and stirred at room temperature. After3-4 hours the reaction mixture was cloudy and after one day a whiteprecipitate formed. After 7 days the reaction mixture was diluted with 5ml of methanol and added to diethylether (100 ml) to form a whiteprecipitate. The solid ψ-brene was collected by centrifugation.Purification of the solid by addition of methanol (5 ml) followed byprecipitation by diethylether (100 ml) was repeated two more times. Aportion of the solid was chromatographed on Sephadex G25 with 0.02Mammonium carbonate buffer.

Method B: Diaminedisulfide (1) (1.04 g, 5 mmol) and 1,3-dibromopropane(1.03 g, 5 mmol) were added to about 4 ml DMSO-H₂ O (75:25 v/v). Thereaction mixture was stoppered and stirred at room temperature. Thereaction mixture was cloudy and clarified in 3-4 hours. A total of 20 mlof water was added periodically over the next eleven days in proportionsto bring the reaction to the cloud point. Thereafter, the water wasevaporated. The product was precipitated using diethylether and aportion of the solid was chromatographed as in Method A.

NMR for Method A and Method B (D₂ O) δ2.50 and 2.55 (small singlets,--N(CH₃)₂ terminal groups), 3.3 (br s, ⁺ NCH₃) and 3.35-4.1 (m, CH₂)ppm.

                  TABLE 1                                                         ______________________________________                                        COPOLYMER           A       B    C    D    E                                  ______________________________________                                        1,6-bisdimethyl-                                                                          (mg)    68.9    172.3                                                                              344.6                                                                              517.0                                                                              620.4                              aminohexane (mmol)  0.4      1.0  2.0  3.0 3.6                                diaminedisulfide(1)                                                                       (mg)    750.2   625.2                                                                              416.8                                                                              208.4                                                                              83.4                                           (mmol)  3.6      3.0  2.0  1.0 0.4                                1,3-dibromopropane                                                                        (mg)    824.0   824.0                                                                              824.0                                                                              824.0                                                                              824.0                                          (mmol)  4.0      4.0  4.0  4.0 4.0                                ______________________________________                                    

EXAMPLE 3 Preparation of Copolymers

Copolymers of the diaminedisulfide (1) and 1,6-dimethylaminohexane with1,3-dibromopropane were prepared in DMSO as specified in Table 1. Thereactions and product isolations were carried out as in Example 1 exceptthat ethylacetate was substituted for diethylether for the first twoprecipitations. Copolymers A and B were white powders; Copolymers C andD were gummy solids and Colpolymer E was a hygroscopic solid. Under highvacuum Copolymers C and D became glassy foams.

EXAMPLE 4 Preparation of Hydroxypolybrene

A mixture of 1,6-bisdimethylaminohexane (1.24 g, 5 mmol),DL-1,4-dibromo-2,3-butanediol (1,24 g, 5 mmol), and DMSO (3.5 ml) wasstoppered and stirred. The initial two-phase mixture became homogeneousafter 3 days. After 6 days the reaction mixture was added to 150 ml ofanhydrous diethylether to give hydroxypolybrene as a gummy oil. Aportion was further purified by dissolution in four times its weight inmethanol, precipitation at -78°, and decantation of the coldsupernatant.

NMR (methanol-d₄): δ0.3-2.3 (m, 8H, --NCH₂ CH₂ CH₂ CH₂ CH₂ --); 2.9 (s,<1H, terminal N(CH₃)₂), 3.3 (br s, 12H, --⁺ N(CH₃)₂ --) 3.4-3.8 (m, 8H,--⁺ N(CH₃)₂ CH₂ --), and 4.3-4.6 (m, 2H, CHOH) ppm.

Addition of acid shifted the terminal dimethylamine singlet downfield.

EXAMPLE 5 Preparation of Polymer from the Disulfide of2-dimethylaminoethanethiol and 1,4-dibromobutane

A solution of diaminedisulfide (1) (834 mg, 4.0 mmol) and1,4-dibromobutane (864 mg, 4.0 mmol) in DMSO (2.8 ml) was stoppered andstirred. After 7 days the pasty mixture was diluted with methanol (5ml), precipitated by dropping the resulting suspension into ethylacetate (100 ml) and collected by centrifugation. The suspension,precipitation and centrifugation cycle was repeated two more times togive a white powder that was dried under vacuum.

EXAMPLE 6 Aggregation of Latex Particles by Polybrene and ψ-Brene

Test mixtures containing latex particles (0.88 mg/ml), ammoniumcarbonate buffer, and varying concentrations of an aggregating agent(Polybrene, ψ-brene, or hydroxypolybrene) were prepared and examinedvisually for aggregation after 0-60 seconds. Mixtures showing noaggregation were reexamined after 4-6 hours. Test mixtures containingclearly discernible aggregates were designated positive (+). Thoseremaining turbid with no discernible aggregates or settling after 6hours were designated negative (-). Mixtures containing a few aggregatesin a cloudy suspension and those containing cloudy suspension thatsettled in 6 hours were designated borderline (±).

Both high and low molecular weight column fractions of ψ-brene fromExample 2 were tested. The disulfide of 2-dimethylaminoethanethiol(diaminedisulfide (1)) was used as a control. Results appear in Table 2below.

                  TABLE 2                                                         ______________________________________                                                          ψ-brene                                                 Concentration     high    low                                                 in the test                                                                             Poly-   mol.    mol.  hydroxy-                                                                              diamine                               mixture   brene   wt.     wt.   polybrene                                                                             control                               ______________________________________                                        0 mg/ml   -       -       -     -       -                                     0.025 mg/ml                                                                             -       -       -     -       -                                     0.050 mg/ml                                                                             +       -       -     -       -                                     0.100 mg/ml                                                                             +       ±    +     +       -                                     0.200 mg/ml                                                                             -       +       +     +       -                                     0.400 mg/ml                                                                             -       +       +     +                                             0.800 mg/ml                     -                                             ______________________________________                                    

The data in Table 2 show that all of the polymers tested causedaggregation of the latex particles. In each case the range ofconcentration of polymer that caused aggregation was at least a factorof two. Diaminedisulfide (1) control did not cause latex particleaggregation.

EXAMPLE 7 Prevention of ψ-Brene-dependent Aggregation

Test mixtures of latex particles (2.2 mg/ml) and DTE (1.22 mM) inammonium carbonate buffer were treated with solutions of ψ-brene inammonium carbonate buffer. The final mixture contained latex particles(0.88 mg/ml), DTE (0.49 mM) and ψ-brene (0.2 mg/ml). Bothhigh-molecular-weight and low-molecular-weight column fractions ofψ-brene from Example 2 were tested. Polybrene (0.1 mg/ml in the finalmixture) was used in place of ψ-brene as a control. Aggregation wasdetected visually as in Example 6.

The presence of DTE in the test mixtures prevented aggregation of latexparticles by ψ-brene. This was observed for both high-molecular-weightand low-molecular-weight fractions of ψ-brene. Aggregation of latexparticles by Polybrene was not prevented by addition of DTE.

EXAMPLE 8 Reversal of ψ-Brene-dependent Aggregation

Aggregated test mixtures (500 ml) containing latex particles (0.88mg/ml) and ψ-brene (0.2 mg/ml) in ammonium carbonate buffer were treatedwith aqueous DTE (10 μl of 25 mM). Both high-molecular-weight andlow-molecular-weight fractions of ψ-brene from Example 2 were tested.Aggregated test mixtures containing Polybrene (0.1 mg/ml) instead ofψ-brene were used as controls. Aggregation was evaluated as in Example6.

Dispersal of the latex particles aggregated with ψ-brene by DTE wasseen. Substitution of H₂ O for DTE did not produce aggregate dispersal.The procedure was repeated substituting Polybrene for ψ-brene. NeitherDTE nor H₂ O produced dispersal of latex particles aggregated byPolybrene.

EXAMPLE 9 Prevention of Hydroxypolybrene-dependent Aggregation

Test mixtures containing latex particles (2.2 mg/ml) and NaIO₄ (5 mM,2.5 mM, 1.25 mM, 0.625 mM or 0 mM) in ammonium carbonate buffer weretreated with hydroxypolybrene in ammonium carbonate buffer such that thefinal mixture contained latex particles (0.88 mg/ml), hydroxypolybrene(0.1 mg/ml) and NaIO₄ (2 mM, 1 mM, 0.5 mM, 0.25 mM, or 0 mM). Two setsof controls were done. In the first set NaIO₄ was replaced by anequivalent concentration of NaCl. In the second set hydroxypolybrene wasreplaced by Polybrene (0.1 mg/ml). Aggregation was evaluated visually asin Example 6.

A NaIO₄ concentration of 2 mM prevented immediate aggregation, whereasconcentrations of 1 mM and 0.5 mM were initially borderline. After 16hours mixtures containing greater than 0.5 mM NaIO₄ were no longeraggregated. Controls without NaIO₄ aggregated immediately and did notchange, as did controls containing Polybrene.

EXAMPLE 10 Reversal of Hydroxypolybrene-dependent Aggregation

Aqueous NaIO₄ (10 μl, 5 μl, 1.5 μl, or 1.2 μl of 0.1M) was added to testmixtures (500 μl) and hydroxypolybrene (0.1 mg/ml) in ammonium carbonatebuffer. Two sets of controls were done. In the first set NaIO₄ wasreplaced by NaCl of equal concentration. In the second sethydroxypolybrene was replaced by Polybrene. Mixtures were examinedvisually for aggregation as in Example 6.

The aggregated latex particles containing hydroxypolybrene weredispersed immediately by the two higher NaIO₄ concentrations. After 16hours all four NaIO₄ concentrations tested had caused dispersal of theaggregated latex particles, whereas without NaIO₄ the aggregatesremained. Controls with NaCl and controls with Polybrene remainedaggregated.

EXAMPLE 11 Aggregation of Blood by ψ-Brene and Copolymers

Blood (10 μl), LISS (85 μl), and a solution of the test polymer (15 μl)in LISS were mixed and evaluated visually for aggregation after 30seconds as in Example 6. The polymers, their concentrations, and theaggregation performance appear in the Table 3 below. Copolymers werefrom Example 3 and ψ-brene was from Example 2.

                  TABLE 3                                                         ______________________________________                                        Copolymer D Copolymer A   ψ-Brene                                         final   Aggre-  final     Aggre-                                                                              final    Aggre-                               concent.                                                                              gation  concent.  gation                                                                              concent. gation                               ______________________________________                                        1.4 mg/ml                                                                             +       1.4 mg/ml +     1.4 mg/ml                                                                              +                                    0.68 mg/ml                                                                            +       0.14 mg/ml                                                                              +     0.68 mg/ml                                                                             +                                    0.14 mg/ml                                                                            -       1.4 μl.ml                                                                            -     0.014 mg/ml                                                                            -                                    0 mg/ml -       0 mg/ml   -     0 mg/ml  -                                    ______________________________________                                    

EXAMPLE 12 Prevention of Red Cell Aggregation

Test mixtures containing washed red blood cells (10 μl of 50% cells inLISS), aqueous DTE (5 μl of 25 mM) and LISS (95 μl) were prepared.Controls were prepared in which DTE was replaced by H₂ O alone. Anaqueous solution of ψ-brene (5 μl of 10 mg/ml) was added to each testmixture and control. Aggregation was evaluated visually as in Example 6after 30 seconds.

The controls containing no DTE aggregated immediately, whereasaggregation was prevented in the test mixture containing DTE.

Red cell agglutination by Polybrene in a similar test is not affected byDTE.

EXAMPLE 13 Reversal of Red Cell Aggregation

Aggregated test mixtures of red cells were prepared by combining redblood cells (10 μl of 50% cells in LISS), LISS (95 μl) and an aqueoussolution of ψ-brene (5 μl of 10 mg/ml). To the test mixtures was addedaqueous DTE (5 μl of 25 mM). In a set of controls H₂ O alone replacedthe aqueous DTE. Aggregation was evaluated visually as in Example 6after 30 seconds.

The test mixtures treated with DTE showed dispersal of the aggregatedcells. The controls without DTE remained aggregated.

Red cells aggregated by Polybrene in a comparable experiment are notdispersed by DTE.

EXAMPLE 14 Preparation of Succinylated Magnetic Particles

Two hundred (200) mg of magnetic particles (Advanced Magnetic, BioMag4100, 4 ml) were washed by magnetic separation (3×40 ml 0.1M phosphatebuffer, pH 7.0) and resuspended in 15 ml of the above buffer. Theparticles were reacted with succinic anhydride (5 ml of 1M in DMF) byaddition of 5 aliquots over 2 hours (the pH was adjusted to 7.0following each addition). The succinylated particles were washed bymagnetic separation (3×40 ml 0.1M phosphate buffer, pH 7.0, and 2×40 mlLISS), resuspendend in 20 ml of LISS and stored at 4° C. with 0.02%sodium azide.

EXAMPLE 15 Whole Blood Separation

Whole blood (480 μl) was mixed with a solution of ψ-brene (80 μl of 20mg/ml in LISS). A suspension of magnetic particles as prepared inExample 14 (2 mg in 248 ml of LISS) was added and the suspension mixed.The aggregates were separated magnetically. Separation was followedvisually as the aggregated cells and magnetic particles were drawntoward the magnets. After 1 minute clear plasma was withdrawn by pipet.

Separation did not occur when ψ-brene was replaced by LISS alone.

EXAMPLE 16 Aggregation, Prevention and Reversal of Blood withHydroxypolybrene

A. Aggregation

Blood (10 μl), LISS (95 μl), and a solution of hydroxypolybrene (5 μl of100 mg/ml, 10 mg/ml, 1 mg/ml, or 0 mg/ml) in LISS were mixed andevaluated after 30 seconds for aggregation as in Example 6. Aggregationwas observed with the two highest hydroxypolybrene concentrationstested.

B. Prevention

Blood (10 μl), LISS (95 μl), and aqueous NaIO₄ (5 μl of 25 mM) werecombined in each test mixture. A solution of hydroxypolybrene (5 μl of10 mg/ml) in LISS was added. Controls in which the NaIO₄ solution wasreplaced by H₂ O were also done. Aggregation was evaluated visuallyafter 30 seconds as in Example 6. No aggregation was seen when NaIO₄ waspresent. Aggregation was observed in the controls lacking NaIO₄.

Aggregation by Polybrene in a comparable experiment is not affected byNaIO₄.

C. Reversal

Aggregated test mixtures were prepared by combining blood (10 μl), LISS(95 μl) and a solution of hydroxypolybrene (5 μl of 10 mg/ml) in LISS.Aqueous NaIO₄ (5 μl of 25 mM) was added to each test mixture. Controlsin which the NaIO₄ solution was replaced by H₂ O alone were also done.Aggregation was evaluated visually after 30 seconds as in Example 6.

Aggregated cells were dispersed in the test mixtures containing NaIO₄.They were not dispersed in the controls that lacked NaIO₄.

In a comparable experiment, cells aggregated by Polybrene are notdispersed by NaIO₄.

The invention has been described in detail with particular reference tothe above embodiments. It will be understood, however, that variationsand modifications can be effected within the spirit and scope of theinvention.

What is claimed is:
 1. A polycation of the formula: ##STR4## wherein R₁and R₂ are the same or different and are independently selected from thegroup consisting of aryl, aralkyl, alkyl, alkylene, alkoxyalkyl,substituted aryl, substituted aralkyl, substituted alkyl, substitutedalkylene, and substituted alkoxyalkyl with the proviso that the "alkoxy"of said alkoxyalkyls has from 1 to 6 carbon atoms and the "alkyl" ineach of the above has from 1 to 6 carbon atoms;B is independentlyselected from the group consisting of --(CH₂)_(a) --(S--S)_(b)--(CH₂)_(c) -- wherein b is 1, a and c are 2 to 8, and n is an averageof 10 to 10,000.
 2. The polycation according to claim 1;wherein R₁ andR₂ are independently selected from the group consisting of alkyl groupsof from one to four carbon atoms; B is independently selected from thegroup consisting of --(CH₂)_(a) --(S--S)_(b) --(CH₂)_(c) -- wherein b is1, a and c are 2 to 8, and n is an average of 10-20.
 3. The polycationaccording to claim 2, wherein R₁ and R₂ are methyl.